Probing nanosecond protein motions of calmodulin by single-molecule fluorescence anisotropy

We report a single-molecule fluorescence anisotropy study of calmodulin, a regulatory protein for calcium-dependent cell signaling. Calmodulin in this study contains a site-specifically inserted tetra-cysteine motif that reacted with FlAsH, a biarsenic fluorescein derivative that can be rotationally locked to the host protein. A photon time-stamping technique was employed that combined the capability for both subnanosecond time resolution of time-correlated single photon counting and single-molecule time trajectory recording. The study provided direct characterization of the nanosecond motions of calmodulin tethered to a biologically compatible surface under physiological buffer solution. The unique technical approaches are applicable to single-molecule study of protein conformational dynamics and protein-protein interactions at a wide range of time scales and without the signal convolution of probe-dye molecular motions. (C) 2004 American Institute of Physics.